Antimicrobial compositions

ABSTRACT

The present invention relates to an enzymatic method for killing or inhibiting microbial cells or microorganisms, e.g. in laundry, on hard surfaces, in water systems, on skin, on teeth or on mucous membranes. The present invention also relates to the use of said enzymatic composition for preserving food products, cosmetics, paints, coatings, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims, under 35 U.S.C. 119, priority or thebenefit of Danish application no. PA 2000 00755 filed May 8, 2000 andU.S. application no. 60/204,710 filed May 16,2000, the contents of whichare fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to an enzymatic antimicrobialmethod for killing or inhibiting microbial cells or microorganismspresent, e.g., in laundry, on hard surfaces, in water systems, on skin,on teeth or on mucous membranes. The present invention also relates tothe use of an enzymatic antimicrobial composition for preserving foodproducts, cosmetics, paints, coatings, etc.

BACKGROUND

[0003] Various enzymatic antimicrobial compositions are known in theart. For instance, WO 94/04127 discloses stabilized dentifricecompositions which are capable of producing antimicrobially effectiveconcentrations of hypothiocyanite ions. The compositions contain anoxidoreductase capable of producing hydrogen peroxide and a peroxidaseenzyme capable of oxidizing thiocyanate ions normally present in salivato antimicrobial hypothiocyanite ions. Suitable peroxidases includelactoperoxidase, myeloperoxidase, salivary peroxidase andchloroperoxidase.

[0004] The object of the present invention is to provide an enhancedmethod for killing or inhibiting microbial cells.

SUMMARY OF THE INVENTION

[0005] According to the present invention there is provided a method forkilling or inhibiting microbial cells comprising treating said microbialcells with a phenol oxidizing enzyme system and an enhancing agentselected from the group consisting of:

[0006] in which C, D, and E independently of each other are:

[0007] and R1, R2, R3, R4, R5, R6, R7, R10, R11, R12, R13, R14, R15,R16, R17 independently of each other are H, OH, C₁₋₈-alkyl, acyl, SO₃H,NO₂, CN, Cl, Br, F, NHR8, N(R8)₂, OR9, C₁₋₈-alkyl-OR9, orC₁₋₈-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkyl or acyl; and X is asingle bond or NH, NCH₃, NC₂H₅, O, S, N═N, CH═N, or CH═CH.

[0008] A and B are 6 membered aromatic rings, and may independently ofeach other be substituted with H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN,Cl, Br, F, NHR8, N(R8)₂, OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9;wherein R8 and R9 are H, C₁₋₄-alkyl or acyl.

[0009] One or more carbon atoms of the aromatic rings of A, B, C, D, andE may independently of each other be substituted with N or S, thusrendering said aromatic ring a heterocyclic ring.

[0010] In further aspects, the present invention relates to methods forkilling or inhibiting microbial cells in laundry, in cosmetic productsor on hard surfaces.

[0011] In still further aspects, the present invention relates to use ofan enzymatic antimicrobial composition for cleaning of contact lenses,for cleaning of water systems, for preserving of paint, and in acleaning-in-place system.

DETAILED DESCRIPTION

[0012] In the context of the present invention the term “antimicrobial”is intended to mean that there is a bactericidal and/or a bacteriostaticand/or fungicidal and/or fungistatic effect and/or a virucidal effectand/or a sporicidal effect, wherein

[0013] The term “bactericidal” is to be understood as capable of killingbacterial cells.

[0014] Bactericidal activity is measured as a logarithmic reduction (logreduction) in the number of living cells or Colony Forming Units pr. ml(CFU/ml), e.g. 1 log reduction corresponds to a reduction in the numberof living cells of Pseudomonas putida ATCC12633 from Y×10^(X) CFU/M(CFU: Colony Forming Units; M: ml or g) to Y×10^(X-1) CFU/M, where X canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, and Y can be any number from 0to 10. The number of living cells are to be determined as the number ofPseudomonas putida ATCC12633, which can grow on LB Agar (#0285, Merck,Germany) plates at 30° C.

[0015] The term “bacteriostatic” is to be understood as capable ofinhibiting bacterial growth, i.e. inhibiting growing bacterial cells.

[0016] The term “fungicidal” is to be understood as capable of killingfungal cells.

[0017] The term “fungistatic” is to be understood as capable ofinhibiting fungal growth, i.e. inhibiting growing fungal cells.

[0018] The term “virucidal” is to be understood as capable ofinactivating virus.

[0019] The term “sporicidal” is to be understood as capable ofinactivating spores.

[0020] The term “microbial cells” denotes bacterial or fungal cells, andthe term “microorganism” denotes a fungus (including yeasts) or abacterium.

[0021] In the context of the present invention the term “inhibitinggrowth of microbial cells” is intended to mean that the cells are in thenon-growing state, i.e., that they are not able to propagate.

[0022] The “phenol oxidizing enzyme system” describes an enzymepossessing peroxidase activity together with a hydrogen peroxide source,or a laccase or laccase related enzyme together with oxygen.

[0023] The term “hard surface” as used herein relates to any surface,which is essentially non-permeable for microorganisms. Examples of hardsurfaces are surfaces made from metal, e.g., stainless steel, plastics,rubber, board, glass, wood, paper, textile, concrete, rock, marble,gypsum and ceramic materials which optionally may be coated, e.g., withpaint, enamel and the like. The hard surface can also be a processequipment, e.g., a cooling tower, an osmotic membrane, a water treatmentplant, a dairy, a food processing plant, a chemical or pharmaceuticalprocess plant. Accordingly, the composition according to the presentinvention is useful in a conventional cleaning-in-place (C-I-P) system.

[0024] Enhancing agent

[0025] The present invention relates to enhancing agents selected fromthe group consisting of:

[0026] in which C, D, and E independently of each other are:

[0027] and R1, R2, R3, R4, R5, R6, R7, R10, R11, R12, R13, R14, R15,R16, R17 independently of each other are H, OH, C₁₋₈-alkyl, acyl, SO₃H,NO₂, CN, Cl, Br, F, NHR8, N(R8)₂, OR9, C₁₋₈-alkyl-OR9, orC₁₋₈-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkyl or acyl; and X is asingle bond or NH, NCH₃, NC₂H₅, O, S, N═N, CH═N, or CH═CH. In apreferred embodiment R1, R2, R3, R4, R5, R6, R7, R10, R11, R12, R13,R14, R15, R16, R17 independently of each other are H, OH, C₁₄-alkyl,acyl, NO₂, Cl, Br, NHR8, N(R8)₂, OR9, C₁₋₄-alkyl-OR9, orC₁₋₄-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkyl or acyl; and X is asingle bond or NH, NCH₃, NC₂H₅, O, S, N═N, CH═N, or CH═CH.

[0028] A and B are six membered aromatic rings, and may independently ofeach other be substituted with H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN,Cl, Br, F, NHR8, N(R8)₂, OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9;wherein R8 and R9 are H, C₁₋₄-alkyl or acyl. In a preferred embodiment Aand B may independently of each other be substituted with H, OH,C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8, N(R8)₂, OR9, C₁₋₄-alkyl-OR9, orC₁₋₄-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkyl or acyl.

[0029] One or more carbon atoms of the aromatic rings of A, B, C, D, E,and F may independently of each other be substituted with N or S, thusrendering said aromatic ring a heterocyclic ring.

[0030] In an embodiment X is a single bond or NH, NCH₃, NC₂H₅, O, S,N═N, CH═N, or CH═CH; preferably X is a single bond or NH, NCH₃, NC₂H₅,O, N═N, CH═N, or CH═CH; preferably X is a single bond or NH, NCH₃,NC₂H₅, S, N═N, CH═N, or CH═CH; more preferably X is a single bond or NH,NCH₃, NC₂H₅, N═N, CH═N, or CH═CH.

[0031] In an embodiment the substituents of A are independently of eachother H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN, Cl, Br, F, NHR8, N(R8)₂,OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9; wherein R8 and R9 are H,C₁₋₄-alkyl or acyl. Preferably the substituents of A are independentlyof each other H, OH, C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8, N(R8)₂, OR9,C₁₋₄-alkyl-OR9, or C₁₋₄-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkylor acyl.

[0032] In an embodiment the substituents of B are independently of eachother H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN, Cl, Br, F, NHR8, N(R8)₂,OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9; wherein R8 and R9 are H,C₁₋₄-alkyl or acyl. Preferably the substituents of B are independentlyof each other H, OH, C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8, N(R8)₂, OR9,C₁₋₄-alkyl-OR9, or C₁₋₄-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkylor acyl.

[0033] In an embodiment R1, R2, R3, R4, and R5 of C are independently ofeach other H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN, Cl, Br, F, NHR8,N(R8)₂, OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9; wherein R8 and R9 areH, C₁₋₄-alkyl or acyl. Preferably R1, R2, R3, R4, and R5 of C areindependently of each other H, OH, C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8,N(R8)₂, OR9, C₁₋₄-alkyl-OR9, or C₁₋₄-alkyl-OOR9; wherein R8 and R9 areH, C₁₋₄-alkyl or acyl.

[0034] In an embodiment R1, R2, R3, R4, and R5 of D are independently ofeach other H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN, Cl, Br, F, NHR8,N(R8)₂, OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9; wherein R8 and R9 areH, CO₁₋₄-alkyl or acyl. Preferably R1, R2, R3, R4, and R5 of D areindependently of each other H, OH, C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8,N(R8)₂, OR9, C₁₋₄-alkyl-OR9, or C₁₋₄-alkyl-OOR9; wherein R8 and R9 areH, C₁₋₄-alkyl or acyl.

[0035] In an embodiment R1, R2, R3, R4, and R5 of E are independently ofeach other H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN, Cl, Br, F, NHR8,N(R8)₂, OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9; wherein R8 and R9 areH, C₁₋₄-alkyl or acyl. Preferably R1, R2, R3, R4, and R5 of E areindependently of each other H, OH, C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8,N(R8)₂, OR9, C₁₋₄-alkyl-OR9, or C₁₋₄-alkyl-OOR9; wherein R8 and R9 areH. C₁₋₄-alkyl or acyl.

[0036] In an embodiment R10, R11, R12, R13, R14, R15, R16 and R17 of Fare independently of each other H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN,Cl, Br, F, NHR8, N(R8)₂, OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9;wherein R8 and R9 are H, C₁₋₄-alkyl or acyl. Preferably R10, R11, R12,R13, R14, R15, R16 and R17 of F are independently of each other H, OH,C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8, N(R8)₂, OR9, C₁₋₄-alkyl-OR9, orC₁₋₄-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkyl or acyl.

[0037] The terms “C_(1-n)-alkyl” wherein n can be from 1 through 8, asused herein, represent a branched or straight, saturated or unsaturatedalkyl group having from one to the specified number of carbon atoms.Typical C₁₋₆-alkyl groups include, but are not limited to, methyl,ethyl, ethenyl (vinyl), n-propyl, iso-propyl, propenyl, isopropenyl,butyl, iso-butyl, sec-butyl, tert-butyl, crotyl, methallyl, pentyl,iso-pentyl, prenyl, hexyl, iso-hexyl and the like.

[0038] The term “acyl” as used herein refers to a monovalent substituentcomprising a C₁₋₆-alkyl group linked through a carbonyl group; such ase.g. acetyl, propionyl, butyryl, isobutyryl, pivaloyl, valeryl, and thelike.

[0039] In an embodiment at least one of the substituents of A are H,preferably at least two of the substituents of A are H, more preferablyat least three of the substituents of A are H, most preferably at leastfour of the substituents of A are H, in particular all the substituentsof A are H.

[0040] In another embodiment at least one of the substituents of B areH, preferably at least two of the substituents of B are H, morepreferably at least three of the substituents of B are H, mostpreferably at least four of the substituents of B are H, in particularall the substituents of B are H.

[0041] In another embodiment at least one of the substituents R1, R2,R3, R4, and R5 of C are H, preferably at least two of the substituentsR1, R2, R3, R4, and R5 of C are H, more preferably at least three of thesubstituents R1, R2, R3, R4, and R5 of C are H, most preferably at leastfour of the substituents R1, R2, R3, R4, and R5 of C are H, inparticular all the substituents R1, R2, R3, R4, and R5 of C are H.

[0042] In another embodiment at least one of the substituents R1, R2,R3, R4, and R5 of D are H, preferably at least two of the substituentsR1, R2, R3, R4, and R5 of D are H, more preferably at least three of thesubstituents R1, R2, R3, R4, and R5 of D are H, most preferably at leastfour of the substituents R1, R2, R3, R4, and R5 of D are H, inparticular all the substituents R1, R2, R3, R4, and R5 of D are H.

[0043] In another embodiment at least one of the substituents R1, R2,R3, R4, and R5 of E are H, preferably at least two of the substituentsR1, R2, R3, R4, and R5 of E are H, more preferably at least three of thesubstituents R1, R2, R3, R4, and R5 of E are H, most preferably at leastfour of the substituents R1, R2, R3, R4, and R5 of E are H, inparticular all the substituents R1, R2, R3, R4, and R5 of E are H.

[0044] In another embodiment at least one of the substituents R10, R11,R12, R13, R14, R15, R16, and R17 of F are H, preferably at least two ofthe substituents R10, R11, R12, R13, R14, R15, R16, and R17 of F are H,more preferably at least three of the substituents R10, R11, R12, R13,R14, R15, R16, and R17 of F are H, more preferably at least four of thesubstituents R10, R11, R12, R13, R14, R15, R16, and R17 of F are H, morepreferably at least five of the substituents R10, R11, R12, R13, R14,R15, R16, and R17 of F are H, more preferably at least seven of thesubstituents R10, R11, R12, R13, R14, R15, R16, and R17 of F are H, mostpreferably at least six of the substituents R10, R11, R12, R13, R14,R15, R16, and R17 of F are H, in particular all the substituents R10,R11, R12, R13, R14, R15, R16, and R17 of F are H.

[0045] In particular embodiments according to the invention theenhancing agent is selected from the group consisting of:

[0046] 4-aminophenol;

[0047] p-Coumaric acid;

[0048] 4,4′-Biphenol;

[0049] 3,3′,5,5′-Tetramethylbenzidine;

[0050] 4,4′-Diaminodiphenylamine sulfate;

[0051] 4,4′-Dimethoxy-N-methyl-diphenylamine;

[0052] 4,4′-Dihydroxydiphenyl ether;

[0053] 4-Hydroxy-4′-dimethylamino azobenzene;

[0054] N′-Benzylidene-N,N-dimethyl-p-phenylenediamine;

[0055] 4-Amino-4′-hydroxystilbene;

[0056] 4′-Bromo-4-(dimethylamino)-chalcone;

[0057] 3-Dimethylamino-9-ethylcarbazole;

[0058] Harmine, HCl H₂O;

[0059] Methylsyringate;

[0060] Propyl sinapate;

[0061] 1,5-Diaminonaphthalene;

[0062] 1,5-Dihydroxynaphthalene;

[0063] 2,7-Dihydroxynaphthalene;

[0064] 7-Methoxy-2-naphthol;

[0065] 2-Hydroxy-1-naphthaldehyde;

[0066] 2-Hydroxy-1-naphthoic acid;

[0067] 8-Aminoquinoline;

[0068] 10-Methylphenoxazine; and

[0069] 3,7-Dibromophenoxazine-10-propionic acid.

[0070] The enhancing agent of the invention may be present inconcentrations of from 1 to 1000 μM, preferably of from 5 to 500 μM, andmore preferably from 10 to 200 μM.

[0071] Hydrogen peroxide/Oxygen

[0072] If the phenol oxidizing enzyme requires a source of hydrogenperoxide, the source may be hydrogen peroxide or a hydrogen peroxideprecursor for in situ production of hydrogen peroxide, e.g.,percarbonate or perborate, or a hydrogen peroxide generating enzymesystem, (e.g., an oxidase together with a substrate for the oxidase,e.g., an amino acid oxidase together with a suitable amino acid), or aperoxycarboxylic acid or a salt thereof. Hydrogen peroxide may be addedat the beginning of or during the process, e.g., typically in an amountcorresponding to levels of from 0.001-25 mM, preferably to levels offrom 0.005-5 mM, and particularly to levels of from 0.01-1 mM.

[0073] If the phenol oxidizing enzyme requires molecular oxygen,molecular oxygen from the atmosphere will usually be present insufficient quantity. If more O₂ is needed, additional oxygen may beadded.

[0074] Phenol Oxidizing Enzyme

[0075] In the context of the present invention the enzyme of the phenoloxidizing enzyme may be an enzyme possessing peroxidase activity or alaccase or a laccase related enzyme.

[0076] The enzyme of the invention may typically be present inconcentrations of from 1 to 100000 μg enzyme protein per liter aqueoussolution, preferably of from 5 to 50000 μg enzyme protein per literaqueous solution, more preferably of from 10 to 10000 μg enzyme proteinper liter aqueous solution, and most preferably of from 50 to 5000 μgenzyme protein per liter aqueous solution.

[0077] Peroxidases and Compounds possessing Peroxidase Activity

[0078] Compounds possessing peroxidase activity may be any peroxidaseenzyme comprised by the enzyme classification (EC 1.11.1.7), or anyfragment derived therefrom, exhibiting peroxidase activity.

[0079] Preferably, the peroxidase according to the invention isproducible by plants (e.g. horseradish or soybean peroxidase) ormicroorganisms such as fungi or bacteria.

[0080] Some preferred fungi include strains belonging to the subdivisionDeuteromycotina, class Hyphomycetes, e.g., Fusarium, Humicola,Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces,Ulocladium, Embellisia, Cladosporium or Dreschlera, in particularFusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii,Myrothecium verrucaria (IFO 6113), Verticillum alboatrum, Verticillumdahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago,Ulocladium chartarum, Embellisia alli or Dreschlera halodes.

[0081] Other preferred fungi include strains belonging to thesubdivision Basidiomycotina, class Basidiomycetes, e.g., Coprinus,Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f.microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaetechrysosporium (e.g. NA-12) or Trametes (previously called Polyporus),e.g., T. versicolor (e.g. PR4 28-A).

[0082] Further preferred fungi include strains belonging to thesubdivision Zygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, inparticular Mucor hiemalis.

[0083] Some preferred bacteria include strains of the orderActinomycetales, e.g. Streptomyces spheroides (ATTC 23965), Streptomycesthermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp.verticillium.

[0084] Other preferred bacteria include Rhodobacter sphaeroides,Rhodomonas palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC15958), Pseudomonas fluorescens (NRRL B-11) and Bacillus strains, e.g.Bacillus pumilus (ATCC 12905) and Bacillus stearothermophilus.

[0085] Further preferred bacteria include strains belonging toMyxococcus, e.g., M. virescens.

[0086] The peroxidase may furthermore be one which is producible by amethod comprising cultivating a host cell transformed with a recombinantDNA vector which carries a DNA sequence encoding said peroxidase as wellas DNA sequences encoding functions permitting the expression of the DNAsequence encoding the peroxidase, in a culture medium under conditionspermitting the expression of the peroxidase and recovering theperoxidase from the culture.

[0087] Particularly, a recombinantly produced peroxidase is a peroxidasederived from a Coprinus sp., in particular C. macrorhizus or C. cinereusaccording to WO 92/16634.

[0088] In the context of this invention, compounds possessing peroxidaseactivity comprise peroxidase enzymes and peroxidase active fragmentsderived from cytochromes, haemoglobin or peroxidase enzymes.

[0089] Determination of Peroxidase Activity (POXU)

[0090] One peroxidase unit (POXU) is the amount of enzyme which underthe following conditions catalyze the conversion of 1 μmole hydrogenperoxide per minute:

[0091] 0.1 M phosphate buffer pH 7.0

[0092] 0.88 mM hydrogen peroxide

[0093] 1.67 mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS)

[0094] 30° C.

[0095] The reaction is followed for 60 seconds (15 seconds after mixing)by the change in absorbance at 418 nm, which should be in the range 0.15to 0.30.

[0096] For calculation of activity is used an absorption coefficient ofoxidized ABTS of 36 mM⁻¹ cm⁻¹ and a stoichiometry of one μmole H₂O₂converted per two μmole ABTS oxidized.

[0097] Laccases and Laccase Related Enzymes

[0098] In the context of this invention, laccases and laccase relatedenzymes comprise any laccase enzyme comprised by the enzymeclassification (EC 1.10.3.2), any catechol oxidase enzyme comprised bythe enzyme classification (EC 1.10.3.1), any bilirubin oxidase enzymecomprised by the enzyme classification (EC 1.3.3.5) or any monophenolmonooxygenase enzyme comprised by the enzyme classification (EC1.14.18.1).

[0099] The above-mentioned enzymes may be microbial, i.e. derived frombacteria or fungi (including filamentous fungi and yeasts), or they maybe derived from plants.

[0100] Suitable examples from fungi include a laccase derivable from astrain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis,Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizoctonia, e.g., R. solani, Coprinus, e.g., C. cinereus,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Pycnoporus, e.g. P. cinnabarinus, Phlebia, e.g., P. radita (WO92/01046), or Coriolus, e.g., C. hirsutus (JP 2-238885).

[0101] Suitable examples from bacteria include a laccase derivable froma strain of Bacillus.

[0102] A laccase derived from Coprinus, Myceliophthora, Polyporus,Pycnoporus, Scytalidium or Rhizoctonia is preferred; in particular alaccase derived from Coprinus cinereus, Myceliophthora thermophila,Polyporus pinsitus, Pycnoporus cinnabarinus, Scytalidium thermophilum orRhizoctonia solani.

[0103] The laccase or the laccase related enzyme may furthermore be onewhich is producible by a method comprising cultivating a host celltransformed with a recombinant DNA vector which carries a DNA sequenceencoding said laccase as well as DNA sequences encoding functionspermitting the expression of the DNA sequence encoding the laccase, in aculture medium under conditions permitting the expression of the laccaseenzyme, and recovering the laccase from the culture.

[0104] Determination of Laccase Activity (LACU)

[0105] Laccase activity is determined from the oxidation ofsyringaldazin under aerobic conditions. The violet colour produced isphotometered at 530 nm. The analytical conditions are 19 mMsyringaldazin, 23 mM acetate buffer, pH 5.5, 30° C., 1 min. reactiontime.

[0106] 1 laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazin per minute at these conditions.

[0107] Determination of Laccase Activity (LAMU)

[0108] Laccase activity is determined from the oxidation ofsyringaldazin under aerobic conditions. The violet colour produced isphotometered at 530 nm. The analytical conditions are 19 mMsyringaldazin, 23 mM Tris/maleate buffer, pH 7.5, 30° C., 1 min.reaction time.

[0109] 1 laccase unit (LAMU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazin per minute at these conditions.

[0110] The composition

[0111] The present invention provides an enzymatic antimicrobialcomposition comprising a phenol oxidizing enzyme system and an enhancingagent of a formula selected from group consisting of:

[0112] as described above.

[0113] The antimicrobial composition according to the invention may beformulated as a solid or a liquid.

[0114] When formulated as a liquid, the composition is typically anaqueous composition.

[0115] When formulated as a solid, the composition is typically apowder, a granulate, a paste or a gelled product.

[0116] It is preferred to use a two-part formulation system in the caseswhere hydrogen peroxide is needed, whereby the hydrogen peroxide isseparate from the other components.

[0117] The composition of the invention may further comprise auxiliaryagents such as wetting agents, thickening agents, buffer, stabilisers,perfume, colorants, fillers and the like.

[0118] Useful wetting agents are surfactants, i.e., non-ionic, anionic,amphoteric or zwitterionic surfactants.

[0119] The composition of the invention may be a concentrated product ora ready-to-use product. In use, the concentrated product is typicallydiluted with water to provide a medium having an effective antimicrobialactivity, applied to the object to be disinfected or preserved, andallowed to react with the microorganisms present.

[0120] The optimum pH of such an aqueous composition is usually acompromise between the optimum stability and optimum activity of theenzyme in question. In one aspect of the invention pH is in the range ofpH 3 to 10.5 (such as pH 4 to 6 or pH 8 to 10), and in another aspect ofthe invention pH is in the range of pH 4 to 10, preferably pH 5 to 9,and more preferably pH 6 to 8.

[0121] In an embodiment, the present invention also provides a medicalcatheter comprising the composition of the invention.

[0122] The method

[0123] The present invention also provides a method for killing orinhibiting microbial cells comprising treating said microbial cells withthe composition of the invention. Said treatment may be carried out withan effective amount of said composition.

[0124] As an “effective amount” is meant an amount suitable forobtaining the required antimicrobial effect in the chosen application;e.g. to reduce the number of living cells to 10%, 1% or less than 1%; orto prevent the number of living cells from doubling during 12 hours, 1day, 5 days, 30 days or more than 30 days.

[0125] The composition of the invention may be capable of reducing thenumber of living cells (killing) of E. coli (DSM1576) to less than 50%(preferably less than 75%, more preferably less than 90%, mostpreferably less than 95%, in particular at least 99%), when incubated 10min. at 20° C. in an aqueous solution containing 1 mg/L of thecomposition.

[0126] The composition may also be capable of increasing the time beforeoutgrowth (inhibition) of E. coli (DSM1576) at 25° C. in a microbialgrowth substrate containing 1 mg/L of the composition by at least 5%,preferably at least 10%, more preferably at least 25%, most preferablyat least 50%, and in particular at least 100%.

[0127] Uses

[0128] The composition of the invention may be incorporated into adetergent or cleaning composition typically comprising other enzymetypes as well (see below).

[0129] The composition of the invention can also be used for inhibitingmicroorganisms present in laundry, by treating the laundry with asoaking, washing or rinsing liquor comprising an effective amount of thecomposition.

[0130] When used for preservation of paint, food, beverages, cosmeticssuch as lotions (e.g. eye lotions), liquids, creams, gels, pastes,ointments (e.g. eye ointments), soaps, shampoos, conditioners,antiperspirants, deodorants, mouth wash, nasal sprays, contact lensproducts, enzyme formulations, or food ingredients, the composition usedin the method of the present invention may be incorporated into e.g.water based paint, unpreserved food, beverages, cosmetics, contact lensproducts, food ingredients or anti-inflammatory product in an amounteffective for killing or inhibiting growth of microbial cells.

[0131] In particular, the composition of the invention may be used as apreservation agent or a disinfection agent in water based paints (seebelow).

[0132] Furthermore, the composition according to the present inventionmay by useful as a disinfectant, e.g., in the treatment of acne,infections in the eye or the mouth, skin infections; in antiperspirantsor deodorants; in foot bath salts; for cleaning and disinfection ofcontact lenses, hard surfaces, teeth (oral care), wounds, bruises andthe like.

[0133] In general the composition of the present invention is useful forcleaning, disinfecting or inhibiting microbial growth on any hardsurface. Examples of surfaces, which may advantageously be contactedwith the composition of the invention are surfaces of process equipmentused, e.g., in dairies, chemical or pharmaceutical process plants, watersanitation systems, paper pulp processing plants, water treatmentplants, and cooling towers. The composition of the invention may be usedin an amount, which is effective for cleaning, disinfecting orinhibiting microbial growth on the surface in question.

[0134] In particular, the composition of the invention may be used fordisinfecting and inhibiting microbial growth in paper and pulpprocessing plants.

[0135] Further, it is contemplated that the composition of the inventioncan advantageously be used in a cleaning-in-place (C.I.P.) system forcleaning of process equipment of any kind.

[0136] The method of the invention may additionally be used for cleaningsurfaces and cooking utensils in food processing plants and in any areain which food is prepared or served such as hospitals, nursing homes,restaurants, especially fast food restaurants, delicatessens and thelike. It may also be used as an antimicrobial in food products and wouldbe especially useful as a surface antimicrobial for cheese, fruits andvegetables and for food in salad bars.

[0137] The composition of the present invention is also useful formicrobial control of water lines, and for disinfection of water, inparticular for disinfection of industrial water.

[0138] In an embodiment, the composition of the present invention isalso useful for treating medical catheters to kill or inhibit microbespresent on the surface of said medical catheters. The composition of theinvention can also be used for treating wounds.

[0139] Conservation/preservation of paints

[0140] Conservation of paint products in cans has in the art beenaccomplished by adding non-enzymatic organic biocides to the paints. Inthe context of the invention paint is construed as a substancecomprising a solid coloring matter dissolved or dispersed in a liquidvehicle such as water, organic solvent and/or oils, which when spreadover a surface, dries to leave a thin colored, decorative and/orprotective coating. Typically isothiazoliones, such as5-chlor-2-methyl-4-thia-zoli-3-on, has been added to the paint asbiocides to inhibit/prevent microbial growth in the paint. The method ofthe invention can however suitably be applied in this field, therebysolving the problem of the ever present environmental bio-hazards ofusing toxic organic biocides by replacing these toxic biocides withenvironmentally compatible enzymes. Thus the present invention providesa method for conservation of a paint comprising contacting said paintwith a phenol oxidizing enzyme and an enhancing agent according to theinvention. Further the invention provides a paint composition comprisinga phenol oxidizing enzyme and an enhancing agent as described in thepresent invention.

[0141] The paint is preferably a water based paint, i.e. the solids ofthe paint is dispersed in an aqueous solution. The paint may contain0-20 % organic solvent, preferable 0-10%, e.g. 0-5%.

[0142] The enzyme may be added to the paint in an amount of 0.0001-100mg active enzyme protein per liter paint, preferably 0.001-10 mg/l, e.g.0.01-5 mg/l, while the enhancing agent may be added in an amount of10-500 μM, preferably 25-250 μM, e.g. 100 μM of the paint composition.

[0143] Detergent Compositions

[0144] The antimicrobial composition of the invention may be added toand thus become a component of a detergent composition.

[0145] The detergent composition of the invention may for example beformulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

[0146] In a specific aspect, the invention provides a detergent additivecomprising the antimicrobial composition of the invention. The detergentadditive as well as the detergent composition may comprise one or moreother enzymes such as a protease, a lipase, a cutinase, an amylase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a laccase, and/or aperoxidase.

[0147] In general the properties of the chosen enzyme(s) should becompatible with the selected detergent, (i.e. pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts. Proteases: Suitableproteases include those of animal, vegetable or microbial origin.Microbial origin is preferred. Chemically modified or protein engineeredmutants are included. The protease may be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-likeprotease. Examples of alkaline proteases are subtilisins, especiallythose derived from Bacillus, e.g., subtilisin Novo, subtilisinCarlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (describedin WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. ofporcine or bovine origin) and the Fusarium protease described in WO89/06270 and WO 94/25583.

[0148] Examples of useful proteases are the variants described in WO92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially thevariants with substitutions in one or more of the following positions:27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218,222, 224, 235 and 274.

[0149] Preferred commercially available protease enzymes includeAlcalase™, Savinase™, Primase™, Duralase™, Esperase™, and Kannase™(Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™,Purafect OXP™, FN2™, and FN3™ (Genencor International Inc.).

[0150] Lipases: Suitable lipases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Examples of useful lipases include lipases from Humicola (synonymThermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described inEP 258 068 and EP 305 216 or from H. insolens as described in WO96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P.pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase,e.g. from B. subtilis (Dartois et al. (1993), Biochemica et BiophysicaActa, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus(WO 91/16422).

[0151] Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

[0152] Preferred commercially available lipase enzymes include Lipolase™and Lipolase Ultra™ (Novozymes A/S).

[0153] Amylases. Suitable amylases (α and/or β) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, α-amylases obtainedfrom Bacillus, e.g. a special strain of B. licheniformis, described inmore detail in GB 1,296,839.

[0154] Examples of useful amylases are the variants described in WO94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially thevariants with substitutions in one or more of the following positions:15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208,209, 243, 264, 304, 305, 391, 408, and 444.

[0155] Commercially available amylases are Duramyl™, Termamyl™,Fungamyl™ and BAN™ (Novozymes A/S), Rapidase ™ and Purastar™ (fromGenencor International Inc.).

[0156] Cellulases: Suitable cellulases include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Suitable cellulases include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g.the fungal cellulases produced from Humicola insolens, Myceliophthorathermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307,U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No.5,776,757 and WO 89/09259.

[0157] Especially suitable cellulases are the alkaline or neutralcellulases having colour care benefits. Examples of such cellulases arecellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO96/29397, WO 98/08940. Other examples are cellulase variants such asthose described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046,U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO98/12307 and PCT/DK98/00299.

[0158] Commercially available cellulases include Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

[0159] Peroxidases/Oxidases: Suitable peroxidases/oxidases include thoseof plant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g. from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

[0160] The detergent enzyme(s) may be included in a detergentcomposition by adding separate additives containing one or more enzymes,or by adding a combined additive comprising all of these enzymes. Adetergent additive of the invention, i.e. a separate additive or acombined additive, can be formulated e.g. as a granulate, a liquid, aslurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

[0161] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated bymethods known in the art. Examples of waxy coating materials arepoly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molarweights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme Ifs preparations may, for instance, be stabilizedby adding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

[0162] The detergent composition of the invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

[0163] The detergent composition comprises one or more surfactants,which may be non-ionic including semi-polar and/or anionic and/orcationic and/or zwitterionic. The surfactants are typically present at alevel of from 0.1 % to 60% by weight.

[0164] When included therein the detergent will usually contain fromabout 1% to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

[0165] When included therein the detergent will usually contain fromabout 0.2% to about 40% of a non-ionic surfactant such as alcoholethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acylN-alkyl derivatives of glucosamine (“glucamides”).

[0166] The detergent may contain 0-65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g. SKS-6 from Hoechst).

[0167] The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

[0168] The detergent may contain a bleaching system, which may comprisea H₂O₂ source such as perborate or percarbonate, which may be combinedwith a peracid-forming bleach activator such astetraacetylethylenediamine or nonanoyloxybenzenesulfonate.Alternatively, the bleaching system may comprise peroxyacids of e.g. theamide, imide, or sulfone type.

[0169] The enzyme(s) of the detergent composition of the invention maybe stabilized using conventional stabilizing agents, e.g., a polyol suchas propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

[0170] The detergent may also contain other conventional detergentingredients such as e.g. fabric conditioners including clays, foamboosters, suds suppressors, anti-corrosion agents, soil-suspendingagents, anti-soil redeposition agents, dyes, bactericides, opticalbrighteners, hydrotropes, tarnish inhibitors, or perfumes.

[0171] The present invention is further illustrated in the followingexamples, which are not in any way intended to limit the scope of theinvention as claimed.

EXAMPLE 1 Antimicrobial Activity of Enhancing Agents

[0172] Screening for enhancing agents with antimicrobial activity wasperformed in 384 well microtiter plates by measuring the ability oflaccase/enhancing agent combinations to inhibit growth of Pseudomonasputida ATCC12633.

[0173] Enhancing agents were dissolved in EtOH at a concentration of 10mM and stored at 4° C. From these stock solutions a 96 well master platecontaining ˜200 μM enhancing agent was generated by adding 6 μl ofenhancing agent stock into 280 μl milliQ water. The layout of the masterplate was as shown in Table 1. TABLE 1 Layout of master plate containingenhancing agent. Columns 1 and 2 contain 50 mM HEPES buffer pH 7.0 andm1-m40 are numbered enhancing agents. 1 2 3 4 5 6 7 8 9 10 11 12 A HepesHepes m1 m1 m9  M9  m17 m17 m25 m25 m33 m33 B Hepes Hepes m2 m2 m10 M10m18 m18 m26 m26 m34 m34 C Hepes Hepes m3 m3 m11 M11 m19 m19 m27 m27 m35m35 D Hepes Hepes m4 m4 m12 M12 m20 m20 m28 m28 m36 m36 E Hepes Hepes m5m5 m13 M13 m21 m21 m29 m29 m37 m37 F Hepes Hepes m6 m6 m14 M14 m22 m22m30 m30 m38 m38 G Hepes Hepes m7 m7 m15 M15 m23 m23 m31 m31 m39 m39 HHepes Hepes m8 m8 m16 M16 m24 m24 m32 m32 m40 m40

[0174]Pseudomonas putida ATCC12633 was grown in a dilution series overnight in LB medium and a late exponential culture was harvested bycentrifugation (5 min @ 4000 RPM in a Microcentrifuge 154, Ole Dich,Denmark) and washed twice in 50 mM HEPES buffer pH 7.0. OD@490 wasadjusted to 0.05 corresponding to approximately 10E5 cells/ml. TABLE 2Layout of screenings plate. Only half of the 384 well plate is shown.Rows 3 5, 7, 9, 11 contain enhancing agent only, whereas rows 4, 6, 8,10, 12 contain enhancing agent and laccase. Rows 1 and 2 are negativecontrols as well as a dilution series of cells are included in order toevaluate the amount of killing observed by the laccase / enhancing agentsystem. All assays were run in duplicate. 1 2 3 4 5 6 7 8 9 10 11 12 AHepes Hepes m1 m1 m9  m9  m17 m17 m25 m25 m33 m33 B Hepes Hepes m1 m1m9  m9  m17 m17 m25 m25 m33 m33 C 10E0 10E0 m2 m2 m10 m10 m18 m18 m26m26 m34 m34 D 10E0 10E0 m2 m2 m10 m10 m18 m18 m26 m26 m34 m34 E 10E110E1 m3 m3 m11 m11 m19 m19 m27 m27 m35 m35 F 10E1 10E1 m3 m3 m11 m11 m19m19 m27 m27 m35 m35 G 10E2 10E2 m4 m4 m12 m12 m20 m20 m28 m28 m36 m36 H10E2 10E2 m4 m4 m12 m12 m20 m20 m28 m28 m36 m36 I 10E3 10E3 m5 m5 m13m13 m21 m21 m29 m29 m37 m37 J 10E3 10E3 m5 m5 m13 m13 m21 m21 m29 m29m37 m37 K Hepes Hepes m6 m6 m14 m14 m22 m22 m30 m30 m38 m38 L HepesHepes m6 m6 m14 m14 m22 m22 m30 m30 m38 m38 M LB LB m7 m7 m15 m15 m23m23 m31 m31 m39 m39 N LB LB m7 m7 m15 m15 m23 m23 m31 m31 m39 m39 O m8m8 m16 m16 m24 m24 m32 m32 m40 m40 P m8 m8 m16 m16 m24 m24 m32 m32 m40m40

[0175] 10 μl of the enhancing agent from the 96 well master plate(Table 1) was transferred to a 384 well plate in duplicate as shown intable 2. The 384 well screening plate was pre-incubated at 40° C. on aheat block (Techne DB-2A, Buch & Holm A/S, Denmark) before the assay wasstarted by addition of 35 μl of preheated buffer (40° C.) containingcells (10E5 CFU/ml) with or without laccase (12,5 μg/ml). The reactionwas incubated for 25 min at 40° C., and 45 μl LB medium was added toallow cells to grow. Growth at 30° C. was monitored using a PolarstarGalaxy microtitter plate reader (BMG Labtechnologies, Germany) in theabsorbance mode. The development in absorbance was measured online for16 hours at 480 nm, and the resulting growth curves were used forevaluation of the anti-microbial activity of the differentlaccase/enhancing agent combinations. The reduction in colony formingunits was estimated by comparison to the standard curve, andanti-microbial enhancing agents were categorized in two groups: Group 1compounds showed between 10E2 and 10E5 reduction and group 2 showed morethan 10E5 reduction (i.e. total kill). Results are shown in tables 3-6.TABLE 3 Enhancing agents with antimicrobial activity mediated by MtL:CFU CAS no. Name Supplier reduction 123-30-8 4-aminophenol

Aldrich >10⁵ 54827-17-7 3,3′,5,5′- Tetramethylbenzidine

Acros 10²-10⁵ 27151-57-1 4,4′-Dimethoxy-N- methyldiphenylamine

Lancaster 10²-10⁵ 1965-09-9 4,4′- Dihydroxyphenyl ether

Sigma- Aldrich (Acros) >10⁵ 2496-15-3 4-Hydroxy-4′- dimethylaminoazobenzene

TCl >10⁵ 3-Dimethylamino-9- ethylcarbazole

Novozymes >10⁵ 884-35-5 Methylsyringate

Lancaster 10²-10⁵ 2243-62-1 1,5- Diaminonaphthalene

Novozymes >10⁵ 582-17-2 2,7- Dihydroxynaphthalene

Novozymes 10²-10⁵ 25782-99-4 10- Methylphenoxazine

Novozymes 10²-10⁵

[0176] TABLE 4 Enhancing agents with antimicrobial activity mediated byRsL: CFU CAS no. Name Supplier reduction 123-30-8 4-aminophenol

Aldrich >10⁵ 92-88-6 4,4′-Biphenol, 97%

Aldrich 10²-10⁵ 6369-04-6 4,4′- Diaminophenylam- ine sulfate, tech. 85%

Janssen Chemica >10⁵ 1965-09-9 4,4′- Dihydroxyphenyl ether

Sigma- Aldrich (Acros) >10⁵ 2496-15-3 4-Hydroxy-4′- dimethylaminoazobenzene

TCl >10⁵ 32-Dimethylamino-9- ethylcarbazole

Novozymes >10⁵ 884-35-5 Methylsyringate

Lancaster 10²-10⁵ Propyl sinapate

Novozymes >10⁵ 2243-62-1 1,5- Diaminonaphthalene

Novozymes >10⁵ 83-56-7 1,5- Dihydroxynaphthalene

Novozymes 10²-10⁵ 582-17-2 2,7- Dihydroxynaphthalene

Novozymes 10²-10⁵ 2283-08-1 2-Hydroxy-1- naphthoic acid

TCl >10⁵ 578-66-5 8-Aminoquinoline

Aldrich >10⁵ 3,7- Dibromophenoxazine- 10-propionic acid

Novozymes >10⁵

[0177] TABLE 5 Enhancing agents with anti-microbial activity mediated byCcL: CFU CAS no. Name Supplier reduction 123-30-8 4-aminophenol

Aldrich >10⁵ 92-88-6 4,4′-Biphenol, 97%

Aldrich 10²-10⁵ 54827-17-7 3,3′,5,5′- Tetramethylbenzidine

Acros 10²-10⁵ 6369-04-6 4,4′- Diaminodiphenylam- ine sulfate, tech. 85%

Janssen Chemica >10⁵ 27151-57-1 4,4′-Dimethoxy-N- methyldiphenylamine

Lancaster 10²-10⁵ 1965-09-9 4,4′- Dihydroxydiphenyl ether

Sigma- Aldrich (Acros) >10⁵ 2496-15-3 4-Hydroxy-4′- dimethylaminoazobenzene

TCl >10⁵ 889-37-2 N′-Benzylidene- N,N-dimethyl-p- phenylenediamine

Sigma- Aldrich 10²-10⁵ 3-Dimethylamino-9- ethylcarbazole

Novozymes >10⁵ 884-35-5 Methylsyringate

Lancaster 10²-10⁵ Propyl sinapate

Novozymes >10⁵ 2243-62-1 1,5- Diaminonaphthalene

Novozymes >10⁵ 83-56-7 1,5- Dihydroxynaphthalene

Novozymes 10²-10⁵ 582-17-2 2,7- Dihydroxynaphthalene

Novozymes 10²-10⁵ 5060-82-2 7-Methoxy-2- naphthol, 98%

Aldrich 10²-10⁵ 2283-08-1 2-Hydroxy-1- naphthoic acid

TCl >10⁵ 578-66-5 8-Aminoquinoline

Aldrich >10⁵ 25782-99-4 10- Methylphenoxazine

Novozymes 10²-10⁵ 3,7- Dibromophenoxazine- 10-propionic acid

Novozymes >10⁵ 3-(3- Hydroxyphenyl)rhod- anine

Sima- Aldrich 10²-10⁵

[0178] TABLE 6 Enhancing agents with anti-microbial activity mediated byPpL: CFU CAS no. Name Supplier reduction 123-30-8 4-aminophenol

Aldrich >10⁵ 92-88-6 4,4′-Biphenol, 97%

Aldrich 10²-10⁵ 54827-17-7 3,3′,5,5′- Tetramethylbenzidine

Acros 10²-10⁵ 6369-04-6 4,4′- Diaminophenylam- ine sulfate, tech. 85%

Janssen Chemica >10⁵ 27151-57-1 4,4′-Dimethoxy-N- methyldiphenylamine

Lancaster 10²-10⁵ 1965-09-9 4,4′- Dihydroxyphenyl ether

Sigma- Aldrich (Acros) >10⁵ 2496-15-3 4-Hydroxy-4′- dimethylaminoazobenzene

TCl >10⁵ 889-37-2 N′-Benzylidene- N,N-dimethyl-p- phenylenediamine

Sigma- Aldrich 10²-10⁵ 836-44-2 4-Amino-4′- hydroxystilbene

Lancaster 10²-10⁵ 3-Dimethylamino-9- ethylcarbazole

Novozymes >10⁵ 884-35-5 Methylsyringate

Lancaster 10²-10⁵ 2243-62-1 1,5- Diaminonaphthalene

Novozymes >10⁵ 83-56-7 1,5- Dihydroxynaphthalene

Novozymes 10²-10⁵ 582-17-2 2,7- Dihydroxynaphthalene

Novozymes 10²-10⁵ 5060-82-2 7-Methyl-2- naphthol, 98%

Aldrich 10²-10⁵ 25782-99-4 10- Methylphenoxazine

Novozymes 10²-10⁵ 3,7- Dibromophenoxazine- 10-propionic acid

Novozymes 10²-10⁵ 3-(3- Hydroxyphenyl)rhod- anine

Sima- Aldrich 10²-10⁵ p-curmaric acid

Novozymes 10²-10⁵

[0179] Reagents

[0180] 50 mM HEPES buffer (Sigma Catalogue # H3375) pH 7.00

[0181] LB Bouillon was from Merck, #0285. 25 g mix was added to 1000 mlwater and then autoclaved.

[0182] The laccases used are described in: Myceliophthora thermophila(MtL) WO 95/33836 Rhizoctonia solani (RsL) WO 95/07988 Coprinus cinereus(CcL) WO 97/08325 Polyporus pinsitus (PpL) WO 96/00290

[0183] which are hereby incorporated by reference.

[0184] Microtiter plates (96 well and 384 well plates) were obtainedfrom Nalge Nunc International (Denmark). Enhancing agents were suppliedas indicated in Table 3-6.

EXAMPLE 2 Antimicrobial Activity of Mediators Oxidized by Peroxidose

[0185] Antibacterial activity of 4-aminophenol,4-hydroxy-4′-dimethylamino azobenzene, 4,4′-biphenol,10-methylphenoxazine and 4,4′-dihydroxydiphenyl ether was evaluated whenoxidized by Coprinus cinereus peroxidase (rCiP) (available fromNovozymes A/S).

[0186] Antimicrobial activity was determined on Pseudomonas putida (ATCC12633), the cells were grown in Tryptone Soy Broth, TSB (Oxoid CM129)overnight, washed two times in sterile 0.9% saline and suspended in 50mM HEPES buffer (Sigma H3375) at pH 7 to a cell concentration ofapproximately 10⁶ CFU/ml. The mediators were added to the cellsuspension to a final concentration of 50 μM, rCiP was added to theconcentration 4 POXU/L and the enzyme reaction was started by additionof hydrogen peroxide to a final concentration of 0.5 mM. Antimicrobialactivity was determined after 20 minutes incubation at 40° C., by a10-fold dilution of the cell suspension into TSB substrate andincubation overnight. Antimicrobial activity was determined as log₁₀reduction, thus a log₁₀ reduction of 2 corresponds to a kill of 99%.TABLE 7 Antimicrobial activity of mediators oxidized by peroxidase.Mediator Log₁₀ reduction 4-amino phenol 2.5 4-hydroxy-4′-dimethylaminoazobenzene 6*  4,4′biphenol 1.5 10-methylphenoxazine 3  4,4′-dihydroxydiphenyl ether 2.5

[0187] None of the evaluated mediators showed any antimicrobial activitywithout rCiP or when combined with hydrogen peroxide.

1. An enzymatic antimicrobial composition comprising a phenol oxidizingenzyme system and an enhancing agent of a formula selected from thegroup consisting of:

in which C, D, and E independently of each other are:

R1, R2, R3, R4, R5, R6, R7, R10, R11, R12, R13, R14, R15, R16, R17independently of each other are H, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN,Cl, Br, F, NHR8, N(R8)₂, OR9, C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9;wherein R8 and R9 are H, C₁₋₄-alkyl or acyl; X is a single bond or NH,NCH₃, NC₂H₅, O, S, N═N, CH═N, or CH═CH; and A and B are six memberedaromatic rings, and are independently of each other be substituted withH, OH, C₁₋₈-alkyl, acyl, SO₃H, NO₂, CN, Cl, Br, F, NHR8, N(R8)₂, OR9,C₁₋₈-alkyl-OR9, or C₁₋₈-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkylor acyl.
 2. The composition of claim 1, in which R1, R2, R3, R4, R5, R6,R7, R10, R11, R12, R13, R14, R15, R16, R17 independently of each otherare H, OH, C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8, N(R8)₂, OR9,C₁₋₄-alkyl-OR9, or C₁₋₄-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkylor acyl; and X is a single bond or NH, NCH₃, NC₂H₅, O, S, N═N, CH═N, orCH═CH; and A and B are independently of each other be substituted withH, OH, C₁₋₄-alkyl, acyl, NO₂, Cl, Br, NHR8, N(R8)₂, OR9, C₁₋₄-alkyl-OR9,or C₁₋₄-alkyl-OOR9; wherein R8 and R9 are H, C₁₋₄-alkyl or acyl.
 3. Thecomposition of claim 1, in which one or more carbon atoms of thearomatic rings of A, B, C, D, E, and F are substituted with N or S. 4.The composition of claim 1, in which at least one of R1, R2, R3, R4, andR5 is H.
 5. The composition of claim 1, in which at least one of R10,R11, R12, R13, R14, R15, R16, and R17 is H.
 6. The composition of claim1, in which the enhancing agent is selected from the group consistingof: 4-aminophenol; p-Coumaric acid; 4,4′-Biphenol;3,3′,5,5′-Tetramethylbenzidine; 4,4′-Diaminodiphenylamine sulfate;4,4′-Dimethoxy-N-methyl-diphenylamine; 4,4′-Dihydroxydiphenyl ether;4-Hydroxy-4′-dimethylamino azobenzene;N′-Benzylidene-N,N-dimethyl-p-phenylenediamine;4-Amino-4′-hydroxystilbene; 4′-Bromo-4-(dimethylamino)-chalcone;3-Dimethylamino-9-ethylcarbazole; Harmine, HCl H₂O; Methylsyringate;Propyl sinapate; 1,5-Diaminonaphthalene; 1,5-Dihydroxynaphthalene;2,7-Dihydroxynaphthalene; 7-Methoxy-2-naphthol;2-Hydroxy-1-naphthaldehyde; 2-Hydroxy-1-naphthoic acid;8-Aminoquinoline; b 10-Methylphenoxazine; and3,7-Dibromophenoxazine-10-propionic acid.
 7. The composition of claim 1,in which the phenol oxidizing enzyme system is a peroxidase and ahydrogen peroxide source.
 8. The composition of claim 7, wherein theperoxidase is horseradish peroxidase, soybean peroxidase or a peroxidaseenzyme derived from Coprinus, Bacillus, or Myxococcus.
 9. Thecomposition of claim 8, wherein the peroxidase is derived from Coprinuscinereus or Coprinus macrorhizus.
 10. The composition of claim 7,wherein the hydrogen peroxide source is hydrogen peroxide or a hydrogenperoxide precursor.
 11. The composition of claim 1, in which the phenoloxidizing enzyme system is a laccase or a laccase related enzymetogether with oxygen.
 12. The composition of claim 11, in which thelaccase is a microbial laccase.
 13. The composition of claim 12, whereinthe laccase is derived from Coprinus, Myceliophthora, Polyporus,Pycnoporus, Scytalidium or Rhizoctonia.
 14. The composition of claim 13,wherein the laccase is derived from Coprinus cinereus, Myceliophthorathermophila, Polyporus pinsitus, Pycnoporus cinnabarinus, Scytalidiumthermophilum or Rhizoctonia solani.
 15. The composition of claim 1,wherein said composition is an aqueous composition.
 16. The compositionof claim 15, wherein the concentration of the phenol oxidizing enzyme isin the range of from 0.001-10 mg enzyme protein per liter.
 17. Thecomposition of claim 15, wherein the concentration of the enhancingagent corresponds to 1-1000 μM.
 18. The composition of claim 1, whereinsaid composition is a granulate.
 19. A method for killing or inhibitingmicrobial cells comprising treating said microbial cells with thecomposition of claim
 1. 20. A detergent composition comprising asurfactant and the composition of claim
 1. 21. A method of inhibitingmicroorganisms present in laundry, comprising treating the laundry witha soaking, washing or rinsing liquor comprising the composition ofclaim
 1. 22. A method of preserving a cosmetic product, comprisingadding an effective amount of the composition of claim 1 to the cosmeticproduct.
 23. The method of claim 22, wherein the cosmetic product is amouth wash composition, a cosmetic liquid or gel or paste, an eyelotion, an antiperspirant, a deodorant, a nasal spray, an eye ointment,an ointment or cream, a foot bath salt.
 24. Use of the composition ofclaim 1 for cleaning or disinfection of contact lenses.
 25. A method ofcleaning, disinfecting or inhibiting microbial growth on a hard surface,wherein the surface is contacted with the composition of claim
 1. 26.The method of claim 25, wherein the hard surface is a process equipmentsuch as a member of a cooling tower, a water treatment plant, a dairy, afood processing plant, a chemical or pharmaceutical process plant. 27.The method of claim 25, wherein the hard surface is a surface of watersanitation equipment.
 28. The method of claim 25, wherein the hardsurface is a surface of equipment for paper pulp processing.
 29. Use ofthe composition of claim 1 in a cleaning-in-place system.
 30. Use of thecomposition of claim 1 for disinfection of water systems.
 31. The use ofclaim 30 for disinfection of water systems in paper pulp processing. 32.Use of the composition of any of claims 1-6 for preserving paint.